Purified transforming growth factor beta

ABSTRACT

A composition for the promotion of cell proliferation and tissue repair in animals having as active ingredients a TGF- beta  which is activated by either a TGF- alpha  or an EGF or both; and methods for administration. As another embodiment these active ingredients can be admixed with other (secondary) growth factors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of Ser. No. 06/581,021, filed Feb.16, 1984 (now abandoned), which is a divisional of Ser. No. 06/500,833,filed June 3, 1983 (now abandoned), which is a continuation-in-part ofSer. No. 06/468,590, filed Feb. 22, 1983 (now abandoned), which is acontinuation-in-part of Ser. No. 06/423,203, filed Sept. 24, 1982 (nowabandoned).

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to compositions which promote repair of tissue,particularly fibroblast cells, in animals, particularly human beings.This invention further relates to a method of treating wounds by thetopical or systemic administration of the compositions.

2. Description of the Prior Art

There is a continuing need for the promotion of rapid cell proliferationat the site of wounds, burns, diabetic and decubitus ulcers, and othertraumata.

A number of "growth factors" are known, which promote the rapid growthof animal cells. These growth factors include epidermal growth factor(EGF), transforming growth factors (TGF's), and nerve growth factors(NGF). However, prior to this invention, none of these growth factorshave been found to be pharmaceutically acceptable agents for theacceleration of wound healing.

It has been shown that the mitogenic activity of insulin (a hormone) canbe increased many-fold by the presence of prostaglandin F.sub.α (notexactly a hormone, but having similar properties--it causes constrictionof vascular smooth muscle), see L. Jimenez de Asua et al, Cold SpringHarbor Conf. Cell Proliferation, Vol. 6, Sato, ed., Cold Spring HarborLabs., New York (1979) at pp. 403-424. Similar activation of insulin hasbeen reported with fibroblast growth factor by P. S. Rudland et al,Proc. Natl. Acad. Sci. USA, 71, 2600-2604 (1974) and with EGF by R. W.Holley et al, Proc. Natl. Acad. Sci. USA, 71, 2908-2911 (1974).Furthermore, in the "competence-progression" scheme of C. D. Stiles etal, in Proc. Natl. Acad. Sci. USA, 76: 1279-1283 (1979), positiveeffects on cell growth have been demonstrated for platelet-derivedgrowth factor or fibroblast growth factor in combination with members ofthe insulin family such as somatomedins A and C, the insulin-like growthfactors.

Many new peptide growth factors have been isolated and characterizedrecently, as indicated in Tissue Growth Factors, R. Baserga, ed.,Springer-Verlag pub., New York (1981), however there have been fewstudies on the activity of these materials in vivo. In many cases, therelatively small amounts of peptides available have limited the abilityto study their properties in vivo. An important area for potentialapplication of peptide growth factors is the enhancement of woundhealing. Despite the need for rapid wound healing in the treatment ofsevere burns, trauma, diabetic and decubitus ulcers, and many otherconditions, at present there is no practical way to accelerate woundhealing with pharmacological agents. Although it is suggested in TissueGrowth Factors, supra, at p. 123 that EGF might be of benefit in thisarea, it has yet to be extensively used in a practical way for woundhealing.

SUMMARY OF THE INVENTION

This invention affords compositions for the promotion of cellproliferation in animals, especially fibroblast cells in human beings.The compositions have as their active ingredients, beta-typetransforming growth factor (TGF-β) and an activating agent. Theactivating agents of this invention are selected from at least one ofepidermal growth factor (EGF) and alpha-type transforming growth factor(TGF-α).

The TGF-β and the activating agent are preferably present in aboutequimolar amounts, and the active ingredients are present in an amountat least sufficient to stimulate cell proliferation (tissue repair).

As another embodiment, the activated TGF-β compositions of thisinvention may be admixed with other (secondary) growth factors toenhance their activity.

The compositions may be formulated in any suitable carrier for topicalapplication, such as physiological saline solution or purified collagensuspension. The compositions also may be formulated in any suitablecarrier for systemic administration.

The method of topical administration of the compositions of thisinvention is by direct application to a burn, wound, or other traumatasitus. Periodic or continual further administration may be preferablyindicated in most instances, since the active ingredients arephysiologically utilized by the cells whose growth is being stimulated.

As a further embodiment, the compositions of this invention may beadministered systemically by injection, enterally, transdermal patches,and the like, depending upon the nature and site of the traumata to betreated.

It has been discovered that platelets contain 40-100 fold more TGF-βthan do the other non-neoplastic tissues examined to date. Completepurification of this platelet factor now shows that TGF-β is apolypeptide of about 25,000 daltons which is probably composed of two12,500-dalton subunits held together by disulfide bonds. Its molecularweight, subunit structure, and amino acid composition differ from thoseof platelet-derived growth factor (PDGF). In contrast to most growthfactors, platelet-derived TGF-β does not appear to exert its growthpromoting property by directly stimulating total DNA synthesis.

Acidic ethanol extracts of human platelets induced non-neoplasticNRK-fibroblasts to undergo anchorage-independent growth. Less than 100ng/ml of the crude extract elicits 50% of the maximal biologicalresponse when assayed in the presence of epidermal growth factor (2.5ng/ml). In the absence of epidermal growth factor the potency of theextract decreased 1000-fold. These results show that platelets contain atype β transforming growth factor (TGF-β). The specific activity of theplatelet extract is 100-fold greater than that of other non-neoplastictissues. The growth factor was purified to homogeneity by sequential gelfiltration, first in the absence and then in the presence of urea. Theseresults, and its lack of strong mitogenic activity, show that thisprotein is distinct from platelet-derived growth factor. When completelypurified, platelet derived TGF-β elicits 50% of its maximal biologicalresponse at concentrations less than 5×10⁻¹² M.

A polypeptide transforming growth factor (TGF), which inducesanchorage-dependent rat kidney fibroblasts to grow in soft agar, hasbeen isolated from human placenta and purified to homogeneity. Thispolypeptide is classified as a type β TGF because it does not competewith epidermal growth factor (EGF) for membrane receptor sites but doesrequire EGF for induction of anchorage-independent growth of indicatorcells. Purification of this peptide was achieved by acid-ethanolextraction of the placenta, followed by gel filtration, cation-exchangeand high-pressure liquid chromatography of the acid soluble proteins.Homogeneity of the TGF-β from the final column was shown by its constantspecific activity and amino acid composition across the peak of softagar colony forming activity and by its migration as a single band at23,000 to 25,000 molecular weight after NaDodSO₄ -polyacrylamide gelelectrophoresis. Under reducing conditions, the protein migrated on agel as a single band at 13,000 molecular weight. The purified placentalTGF-β caused half maximal growth stimulation of indicator cells in softagar at 64-72 pg/ml (3×10⁻¹² M) in the presence of 2 ng/ml (3.4×10⁻¹⁰ M)of EGF.

DETAILED DESCRIPTION OF THE INVENTION

The term, "transforming growth factor" (TGF) has been defined to includethe set of polypeptides which confer the transformed phenotype onuntransformed indicator cells. The transformed phenotype isoperationally defined by the loss of density-dependent inhibition ofgrowth in monolayer, overgrowth in monolayer, characteristic change incellular morphology, and acquisition of anchorage independence, with theresultant ability to grow in soft agar. Untransformed, non-neoplasticcells will not form progressively growing colonies in soft agar, whilethe property of anchorage-independent growth of cells in culture has aparticularly high correlation with neoplastic growth in vivo.

Although TGFs were first discovered in the conditioned medium ofvirally-transformed neoplastic mouse cells, the application of theacid/ethanol method for extraction of peptides from tissues has nowshown that TGFs can be found in almost all tissues, both neoplastic andnon-neoplastic, from all species of animals that have been examined thusfar. Although TGF activity is usually measured with an in vitrophenotypic transformation assay, this does not imply that TGF activityin vivo is necessarily related to the development of malignancy. Indeed,the transformed phenotype is a physiological state associated withnormal embryological development, and transforming (onc) genes have beenfound in normal cells of essentially all vertebrates. The function ofthese onc genes from normal cells is not known at present. While theremay be irreversible and excessive expression of TGF activity duringmalignant cell growth, the data at hand indicate that TGFs have a morebenign and perhaps essential role in the function of normal cells. Atpresent, it is not known what the intrinsic physiological roles of TGFsare. In this respect, TGFs are like many other peptide hormones orhormone-like agents which have recently been discovered and isolated;this is particularly true for many peptides of the nervous system, forwhich a defined chemical structure may be known, yet whose physiology isstill a matter of uncertainty.

The initial description of sarcoma growth factor (SGF), the first of theTGFs to be isolated, was an important finding in tumor cell biologysince it provided a direct mechanism for the expression of theneoplastic phenotype in a virally-transformed cell. Two importantproperties of SGF were described in these earliest studies, namely (1)that the effects of SGF in causing phenotypic transformation weredependent on its continued presence, and that these effects werereversible when SGF was removed, and (2) that the effects of SGF couldbe expressed in the very same cells that synthesized this peptide, aproperty that has been termed autocrine secretion. Although these twoproperties have not been definitively shown for all of the other morenewly discovered TGFs, the functions of the entire set of TGFs can mostreasonably be assumed to be that of local, hormone-like agents thatreversibly control cell function by paracrine or autocrine mechanisms.

Since the discovery of SGF in 1978, many TGFs have been described fromdiverse sources. These TGFs can be categorized into two groups:extracellular TGFs isolated from conditioned media of cultured cells,and intracellular (cell-associated) TGFs isolated by direct extractionof cells or tissues. Although extracellular TGFs have recently beenisolated from non-neoplastic murine cells, use of conditioned mediumhas, in general, been restricted to neoplastic cell lines that could begrown in long-term, large-scale culture, including certain virally andchemically-transformed rodent cells and human tumor cell lines. Theadaptation of an acid/ethanol extraction procedure to TGF isolationremoved all limitations on cell types and quantities of tissues thatcould be examined. Using this procedure, extracts of all tissues orcells, whether of neoplastic or non-neoplastic origin, whether fromadult or embryonic tissue, whether from human, bovine, or murinegenomes, have been shown to promote colony formation in a soft agarassay; hence, by definition, these extracts contain TGF activity.

A variety of both epithelial and fibroblastic cell lines form coloniesin soft agar in the presence of TGFs. However, the most commonly usedindicator cell line is the rat kidney fibroblast cell clone, NRK 49F,which has been selected for its strong colony-forming response to theTGFs. Rat-1 cells and mouse AKR-2B cells have also been usedsuccessfully as indicator cells.

All of the TGFs referred to above are low molecular weight polypeptideswhich share with SGF the physical properties of acid and heat stabilityand sensitivity to treatment with both trypsin and dithiothreitol.However, there are marked differences in the biological properties ofthese TGFs, particularly with respect to their relationship to EGF.Certain TGFs, though antigenically distinct from EGF, have somestructural homology to EGF, since they compete with EGF for receptorbinding. Other TGFs do not compete with EGF for receptor binding, butinstead are dependent on EGF for activity in the soft agar assay forcolony formation. To remove the ambiguities implicit in the assignmentof the general term "TGF" to these different factors, an operationalclassification of the members of the TGF family based on theirinteractions with EGF is suggested, both with respect to competition forbinding to the EGF receptor and to the requirement for EGF for inductionof colonies in soft agar.

As defined for purposes of this invention, TGF-α are those TGFs whichcompete with EGF for receptor binding and which do not require EGF forthe induction of colony formation in soft agar. TGFs with theseproperties include SGF and other TGFs derived from neoplastic cells, aswell as some TGFs from mouse embryos.

As defined for purposes of this invention, TGF-β are those TGFs which donot compete with EGF for receptor binding and which require EGF for theinduction of colony growth in soft agar. When assayed in the presence ofEGF, TGF-β represents the major colony-forming activity of theintracellular TGFs of both neoplastic and non-neoplastic cell lines andtissues. It can be assumed that TGF-β will be found in conditioned mediaas well, once the proper assays are used.

Those TGFs which do not compete with EGF for receptor binding and whichdo not require EGF for colony formation are designated TGF-γ (gamma-typeTGF). Such TGFs have been described in conditioned media of certainvirally or chemically transformed cells. Finally, TGF-δ (delta-type TGF)is used to specify those TGFs which would both compete for EGF receptorsand require EGF for colony formation in soft agar. EGF itself could beclassified as a weak TGF-δ.

EXAMPLE OF PURIFICATION AND PROPERTIES

Research in our laboratory has been directed toward the isolation ofTGFs directly from cells and tissues. An acid/ethanol extractionprocedure was modified for this purpose, as disclosed in A. B. Robertset al, Proc. Natl. Acad. Sci. USA, 77:3494-3498 (1980), andchromatography and high pressure liquid chromatography (HPLC) have beenemployed for further purification. TGF activity, measured by the abilityto induce non-neoplastic indicator cells (NRK) to form colonies in softagar, has been quantitated on an image analysis system with respect toboth the number and size of the colonies formed. By use of HPLC, we haveshown that two distinctly different TGFs, here classified as TGF-α, andTGF-β, can be isolated from the same pool of acid/ethanol extracts ofMSV-transformed 3T3 cells; for this purpose, columns using a lineargradient of acetonitrile in 0.1% trifluoroacetic acid have been used.TFG-α, which elutes from the column earlier than marker EGF, ischaracterized by its ability to induce the formation of small colonies(850-3,100 μm²) in soft agar in the absence of added EGF and its abilityto compete with EGF in a radio-receptor assay. TGF-β, which elutes laterthan TGF-α or marker EGF, does not compete with EGF for receptor bindingand requires EGF to induce the formation of large colonies (>3,100 μm²)in the soft agar assay.

TGF-α from MSV-transformed 3T3 cells resembles SGF isolated from theconditioned medium of the same cells and other TGFs isolated from ratand human tumor cell lines. Recently, SGF and the TGF-α's from theconditioned media of a human melanoma cell line and virally-transformedrat embryo fibroblasts have been purified to homogeneity. The humanmelanoma TGF-α is a single chain polypeptide of molecular weight 7,400.Its amino acid composition and chromatographic behavior are markedlydifferent from that of human EGF, but similar to that of murine SGF andrat TGF-α, suggesting that TGF-α's from human, rat and mouse genomes aremore closely related to each other than to EGF. There is therefore areasonable possibility that TGF-α's may have cross-species utility.

In sarcoma virus-transformed rodent cell lines, the release of TGF-αinto the medium has been correlated with the expression of thetransformed phenotype, and within a selected set of human tumor celllines that release TGF-α, the ability of the tumor cells to grow in softagar has been correlated with the quantity of TGF-α they secrete.However, secretion of TGF-α is not an absolute requirement forneoplastic behavior; certain chemically transformed murine cell linesand human lung carcinoma cell lines that do not secrete TGF-α releasestrong TGF activity that does not compete with EGF for receptor binding.

TGF-β of the acid/ethanol extract of the MSV-transformed 3T3 cellsresembles other TGFs isolated from many neoplastic and non-neoplastictissues. After further purification on a second HPLC column, TGF-β ofthe MSV-transformed cells eluted at the same position in the n-propanolgradient (48%) as one peak of TGF-β activity of the bovine salivarygland, and each was associated with a small peak of absorbance at 280nm. These two TGF-β's, one from a neoplastic mouse cell line and theother from a non-neoplastic bovine tissue, each migrated as a12,500-13,000 daltons MW protein on SDS-PAGE in the presence ofmercaptoethanol and as an apparent 25,000-26,000 daltons protein in theabsence of mercaptoethanol; they therefore appear to be closely relatedto each other and different from both TGF-α and EGF. The finding ofTGF-β in all non-neoplastic tissues examined thus far suggests a normalphysiological function for these TGFs. There is therefore a reasonablepossibility that TGF-β's may have cross-species utility.

SEQUENCING OF TGF-β

Through a combination of techniques, TGF-β from bovine kidneys waspurified 200,000-fold to the point of homogeneity.

For amino-terminal sequence analysis, approximately 500 picomoles (M_(r)25,000) of TGF-β were reduced and S-carboxymethylated withdithiothreitol and iodo-[¹⁴ C] acetic acid in the presence of 6Mguanidine-HCl in 1M Tris-HCl buffer, pH 8.4. Excess reagents wereseparated from carboxymethylated protein by HPLC on a 5 micron 50×4.6 mmcolumn eluted with a gradient of 0-90% acetonitrile (1% per min) in 0.1%TFA. Overall recovery of the procedure was 96%, based on estimating theamount o protein by amino acid analysis using fluorescamine detection.

Automated Edman degradation was performed on about 500 pmoles (M_(r)12,500) of the S-carboxymethylated protein with a gas-phase sequencer.PTH-amino acids were identified using an HPLC system. Initial yield wasabout 30% and repetitive yield about 90%.

Analysis of the bovine kidney TGF-β by electrophoresis on NaDodSO₄-polyacrylamide gels suggests that some of the disulfide bonds areinterchain.

Amino acid sequence analysis of the reduced and S-carboxymethylatedbovine kidney TGF-β by automated Edman degradation using a gas-phasesequencer revealed a single N-terminal amino acid sequence as follows,(CMC is S-carboxymethylcysteine): ##STR1##

Initial and repetitive yields were found to be equal to the yieldscalculated for myoglobin used as standard protein. At the minimum, theresults indicate that the sequence of at least the first fifteenN-terminal amino acids of each of the two subunits of TGF-β is identicaland confirm the observations of a single protein band of the reducedTGF-β on NaDodSO₄ -polyacrylamide gels. In addition, the N-terminalsequence of the bovine kidney TGF-β is identical to the partial sequenceof TGF-β from human placenta, suggesting a high degree of relatedness oftype β TGFs from different species and different tissue sources.

ACTIVATION OF TGF-β

Recent experiments in our laboratory have shown that either TGF-α or EGFwill activate TGF-β to induce the formation of large colonies in softagar. Purified TGF-β from the MSV-transformed 3T3 cells, assayed byitself, had no colony-forming activity at concentrations as high as 2μg/ml. However, assayed after activation by the presence of either EGF,or TGF-α derived from the same cells, TGF-β induced a dose-dependentformation of large colonies (>3,100 μm²) at concentrations of 10-200ng/ml. By contrast, EGF or TGF-α, assayed by themselves, induced amaximal response of only a small number of colonies; this response wasincreased 10-fold by the addition of TGF-β. The relative abilities ofEGF and TGF-α to promote TGF-β-dependent formation of large colonies insoft agar correlated with their relative abilities to compete forbinding to the EGF receptor; other experiments using chemically-modifiedEGF analogues have substantiated this correlation. These data,demonstrating that the induction of a strong colony-forming responserequires both TGF-α and TGF-β or EGF, suggest that TGF-β, which is foundin all tissues, may be an essential mediator of the effects of TGF-α andof EGF on neoplastic transformation.

Little is known about the mechanisms by which exogenous TGFs inducenon-neoplastic cells to express the transformed phenotype. Furthermore,the synergistic interactions of TGF-α and TGF-β suggest that these twoTGFs may act through different pathways. Experiments using TGFs ofconditioned media of sarcoma virus-transformed rodent cells have shownthat the synthesis of new RNA and protein is required beforetransformation occurs. Other experiments have been directed at apossible role of TGFs in phosphorylation reactions. Certain viraltransforming gene products and their normal cellular homologues havetyrosine-specific protein kinase activity, and it has been proposed thatphosphorylation at tyrosine of specific substrates may be important inthe transformation process. Treatment of human carcinoma A431 cells withvarious TGFs derived from conditioned media of virally-transformed cellsor human tumor cell lines (TGF-α) resulted in phosphorylation oftyrosine residues in the 160K EGF receptor. The pattern ofphosphorylation, however, was indistinguishable from that induced by EGFitself, and thus would not appear to be transformation-specific.Likewise, dissolution of actin fibers of Rat-1 cells occurs when theyare treated with either TGF or EGF. It is clear that further research isneeded to establish the relationships of the TGFs to the retrovirustransforming gene products and the mode of action of the TGFs inneoplastic transformation.

The following are summaries of examples which illustrate various aspectsof this invention:

EXAMPLE 1

HPLC Separation of TGF-α and TGF-β of MSV-Transformed 3T3 Cells. Theacid/ethanol extract of MSV-transformed cells was chromatographed onBio-Gel P-30 in 1M acetic acid. The 7-10,000 MW TGF fraction was furtherpurified on a μBondapak C18 column using a gradient of acetonitrile in0.1% trifluoroacetic acid. Aliquots were assayed for colony-formingactivity in the soft agar assay; in the presence of 2 ng/ml EGF; and incompetition with ¹²⁵ I-EGF in a radio-receptor assay.

EXAMPLE 2

HPLC purification on μBondapak CN columns of TGF-β from MSV-transformedmouse 3T3 cells and bovine salivary gland using a gradient of n-propanolin 0.1% trifluoroacetic acid. TGF-β of acid/ethanol extracts waspurified on Bio-Gel P-30 and μBondapak C18 columns and then applied toCN columns. Aliquots were assayed for induction of colony growth of NRKcells in soft agar in the presence of 2 ng/ml EGF.

EXAMPLE 3

Synergistic interaction (activation) of TGF-β with TGF-α to induce theformation of large colonies of NRK cells in soft agar. Soft agarcolony-forming activity of varying concentrations of μBondapakCN-purified TGF-β derived from MSV-transformed 3T3 cells was assayedeither alone or in the presence of either CN-purified TGF-α derived fromthe same cells or murine EGF. Soft agar colony-forming activity ofvarying concentrations of EGF or TGF-α was assayed either alone or inthe presence of TGF-β.

In Vivo Demonstration of Wound Healing

After the above in vitro demonstrations of the operability of thecompositions of this invention, it was considered critical to confirmthat the compositions could work in clinical applications. For thispurpose, TGFs were isolated on a relatively large scale from bovinesources and the wound healing activity of the compositions according tothis invention was satisfactorily demonstrated using an experimentalrodent wound healing protocol.

The examples which follow demonstrate not only that the compositionsaccording to this invention are effective in vivo, but also that TGFsmay be employed cross-species.

EXAMPLE 4

Purification and separation of TGF-α and TGF-β. Bovine tissues, obtainedfresh from the slaughterhouse and frozen immediately on dry ice, wereextracted in 2 kg batches with acid/ethanol in accordance with A. B.Roberts et al, Pro. Natl. Acad. Sci. USA, 77:3494 (1980). Extracts from6-8 kg tissue were combined and chromatographed on Bio-Gel P-30 with 1Macetic acid, using an 80 liter bed volume column. The TGFs of extractsof bovine kidney or bovine salivary gland eluted in a broad peak betweenthe RNase (13,700) and insulin (5,700) markers, as had been observed forthe TGFs of mouse kidney and mouse salivary gland. TGFs at this stage ofpurification had a specific activity approximately 10 to 25-fold higherthan the acid/ethanol extracts, with a range of recovery of150,000-200,000 colony-forming units per kg tissue. Most of the in vivostudies reported below were done with salivary gland or kidney TGFspurified to this stage. The TGF's activity in vitro was enhancedapproximately 20-fold by the presence of 2-5 ng EGF per ml in the assay,in accordance with this invention.

Following chromatography on Bio-Gel P-30, the bovine TGF-β were purifiedfurther by High Pressure Liquid Chromatography (HPLC) on μBondapak C18columns using an acetonitrile gradient in 0.1 percent trifluoroaceticacid, followed by a second HPLC step on μBondapak CN columns using agradient of n-propanol in 0.1 percent trifluoroacetic acid. After thetwo HPLC steps, analysis of the bovine TGF-βs from both salivary glandand kidney by sodium dodecyl sulfate polyacrylamide gel electrophoresisunder reducing conditions showed a single band with an apparentmolecular weight of 13,000 daltons. At this stage of purification, eachof the bovine TGF-β's had an absolute requirement for EGF forcolony-forming activity. The yield of HPLC-purified TGF-β wasapproximately 20-100 μg per kg tissue, with a total activity of7,000-18,000 colony-forming units.

EXAMPLE 5

Wound healing protocol. In vivo activity of isolated salivary glandTGF-β and kidney TGF-β was measured in accordance with the protocoldescribed by T. K. Hunt et al, Amer. J. Surgery, 114:302 (1967). Sixempty Schilling-Hung wire mesh wound chambers were surgically insertedsubcutaneously in the backs of rats, in paired symmetrical fashion (A-D,B-E, C-F) as shown below:

                  TABLE 1                                                         ______________________________________                                        head                                                                                   A   D                                                                         B   E                                                                         C   F                                                                tail                                                                          ______________________________________                                    

The rats respond to these chambers as if they were wounds, andeventually the chambers become filled with fibroblasts and collagen. Bythe fourth day after insertion, the chambers become encapsulated withconnective tissue, but there are few cells within the chambersthemselves. There is thus a defined, enclosed space within the chambers,where a wound healing response can be quantitatively measured. At thistime, daily injections of TGF-β (0.1 ml, in sterile phosphate-bufferedsaline) into chambers A, B, and C were begun. To activate TGF-βactivity, a low level of murine EGF was included in all TGF-βinjections, unless noted otherwise. Chambers D, E, and F were used ascontrols, and were injected with either an amount of bovine serumalbumin (BSA) alone or in combination with either TGF-β or EGF, suchthat the total protein was equivalent to the amount of TGF-β injectedinto chambers A, B, and C. Injections were made once daily for either 5days (Table 1) or 9 days (Table 3). All injected materials were sterile.The rats were sacrificed 6 hours after the last TGF-β injection; inTable 2 they were injected with 0.5 mCi of thymidine-³ H, specificactivity 6.7 Ci/millimole (i.p.) together with the last TGF-β injection.The chambers were removed from the rats, all connective tissues on theoutside of the wire mesh was peeled away, and then the contents of eachchamber were determined.

                                      TABLE 2                                     __________________________________________________________________________    Wound healing response to bovine salivary gland or kidney TGF after 5         days of treatment.                                                            TGF-βs were prepared and injected as described. Each dose contained      25 times the amount of                                                        TGF-β found optimal for colony formation by NRK cells in a standard      soft agar assay, and ranged                                                   from 18-42 colony forming units per dose. The amounts of protein injected     per dose were: 7 μg                                                        in Expts. 1, 4 and 5; 25 μg in Expt. 3, and 0.7 μg in Expt. 2. All      doses of EGF were 20 ng.                                                      Total protein in wound chambers was measured by the method of Lowry et        al.** Statistical analysis                                                    of the data was made by comparison of matched pairs of the chambers (A        vs. D, B vs. E, C vs. F)                                                      shown in Table 2.                                                                                      Milligrams of                                                                            Average                                   Chamber      Chamber                                                                            Number of                                                                            protein per chamber,                                                                     ratio ±                                A, B, C      D, E, F                                                                            matched pairs                                                                        average (15)                                                                             standard                                  Expt.                                                                             treatment                                                                              treatment                                                                          of chambers                                                                          A, B, C                                                                            D, E, F                                                                             error of mean*                                                                        P                                 __________________________________________________________________________    1   TGF-β (Salivary                                                                   BSA  36     10   3.9   3.8 ± 0.6                                                                          <0.001                                P-30) + EGF                                                               2   TGF-β (Salivary                                                                   BSA  9      8.4  2.9   4.6 ± 1.0                                                                          <0.02                                 HPLC) + EGF                                                               3   TGF-β (Kidney                                                                     BSA  9      8.1  3.5   5.2 ± 1.5                                                                          <0.005                                P-30) + EGF                                                               4   TGF-β (Salivary                                                                   EGF  9      9.6  5.3   2.1 ± 0.3                                                                          <0.02                                 P-30) + EGF                                                               5   TGF-β (Salivary                                                                   TGF-β                                                                         9      11.2 9.6   1.4 ± 0.3                                                                          0.5                                   P-30) + EGF                                                               __________________________________________________________________________     *Average of each matched pair ratio, A/D, B/E, C/F                             One sided .sub.-- P values based on the sign test                            **J. Biol. Chem., 193:265 (1951)                                         

                                      TABLE 3                                     __________________________________________________________________________    Wound healing response to bovine salivary gland TGF-β after 9 days       of treatment.                                                                 Chambers A, B, and C were dosed once daily with 7 μg of TGF-β         (P-30) plus 20 ng of                                                          EGF. Chambers D, E and F were dosed with an equal amount of BSA.                                                 Average                                              Number of                                                                            Average  Average  ratio ±                                           matched pairs                                                                        content per                                                                            content per                                                                            standard                                   Measurement                                                                             of chambers                                                                          chamber A, B, C,                                                                       chamber D, E, F                                                                        error of mean*                                                                        P                                  __________________________________________________________________________    Protein,  30     24       15       1.6 ± 0.05                                                                         <0.001                             milligrams                                                                    DNA,      30     21       8.6      2.6 ± 0.16                                                                         <0.001                             micrograms                                                                    Thymidine-.sup.3 H,                                                                     30     45       30       1.7 ± 0.09                                                                         <0.001                             cpm per microgram                                                             of DNA                                                                        Collagen,  9     5.2      3.2      1.8 ± 0.2                                                                          <0.005                             milligrams                                                                    __________________________________________________________________________     *Average of each matched ratio pair, A/D, B/E, C/F                             One sided .sub.-- P values based on the sign test                       

Table 1 shows that 5 days of treatment of rats with TGF-β from eitherbovine salivary gland or bovine kidney caused a significant increase intotal protein in the treated chambers, as compared to control chamberstreated with an equivalent amount of bovine serum albumin (Experiments1, 3). The salivary gland TGF-β was still highly active after two stepsof purification by the high pressure liquid chromatography (Experiment2). The effects observed are not the sole result of the minute amountsof EGF which had been used to potentiate the activity of TGF-β, since ahighly significant difference between treated chambers A, B and C,compared to control chambers D, E and F was still observed when EGF wasused as the control substance (Experiment 4). Furthermore, when allchambers were treated with TGF-β, and only A, B and C were treated withEGF, no significant difference was observed (Experiment 5). At the endof Experiments 1-4, it was consistently observed that chambers A, B andC were more firmly fixed in the surrounding connective tissue than therespective matched control chambers, suggesting that effects of theTGF-β also were manifested in the area immediately surrounding thechambers.

In order to measure the effects of bovine salivary TGF-β on DNA andcollagen content of the chambers, it was necessary to treat the animalsfor longer than 5 days. Table 2 shows the results of a larger experimentin which 13 rats were treated for 9 days. The increases in totalprotein, total DNA, thymidine incorporation into DNA, and total collagenwere all highly sufficient. Histological examination of the contents ofthe chambers treated with TGF-β confirmed the occurrence of fibroblasticproliferation and formation of collagen. A sterile infiltrate ofinflammatory cells was also found within both treated and controlchambers.

The results obtained in both experiments indicate that TGF-βs whenactivated in accordance with this invention, can significantlyaccelerate a wound healing response.

Clinical Use of the Compositions of This Invention

The compositions of this invention, whose active ingredients are TGF-βactivated by at least one of a TGF-α and an EGF, can reasonably beexpected to have clinical use in the treatment of animals, particularlymammals, most particularly human beings. There are several sound basesfor this conclusion.

It has been shown above, that in in vitro tests, the compositions canmarkedly increase the growth of cells without changing their genotype.An important characteristic of the components of the compositions ofthis invention, is that they do not appear to be species specific. Thatis, TGF-β from one species can be activated by TGF-α and/or EGF fromother species. The cells whose growth is promoted can be of any typesuch as fibroblast or epithelial, although it is considered that thegrowth promotion of fibroblast cells will have the greatest medicalutility.

The in vivo experimental protocol disclosed above, with its veryfavorable results, clearly indicates that the compositions of thisinvention have utility in the treatment of traumata by the rapidpromotion of the proliferation of the cells surrounding the traumata.

Two types of application of the compositions of this invention arecontemplated.

The first, and preferred, application is topically for the promotion ofsurface wound healing. There are no limitations as to the type of woundor other traumata that can be treated, and these include (but are notlimited to): first, second and third degree burns (especially second andthird degree); surgical incisions, including those of cosmetic surgery;wounds, including lacerations, incisions, and penetrations; and surfaceulcers including decubital (bed-sores), diabetic, dental, haemophiliac,and varicose. Although the primary concern is the healing of majorwounds by fibroblast cell regeneration, it is contemplated that thecompositions may also be useful for minor wounds, and for cosmeticregeneration of cells such as epithelial. It is also contemplated thatthe compositions may be utilized by the topical application to internalsurgical incisions.

When applied topically, the compositions may be combined with otheringredients, such as carriers and/or adjuvants. There are no limitationson the nature of such other ingredients, except that they must bepharmaceutically acceptable, efficacious for their intendedadministration, and cannot degrade the activity of the activeingredients of the compositions. When the compositions of this inventionare applied to burns, they may be in the form of an irrigant, preferablyin combination with physiological saline solution. The compositions canalso be in the form of ointments or suspensions, preferably incombination with purified collagen. The compositions also may beimpregnated into transdermal patches, plasters, and bandages, preferablyin a liquid or semi-liquid form.

The second application is systemically for the healing of internalwounds and similar traumata. Such an application is useful provided thatthere are no, or limited, undesirable side-effects, such as thestimulation of neoplastic cellular growth.

When applied systemically, the compositions may be formulated asliquids, pills, tablets, lozenges, or the like, for enteraladministration, or in liquid form for parenteral injection. The activeingredients may be combined with other ingredients such as carriersand/or adjuvants. There are no limitations on the nature of such otheringredients, except that they must be pharmaceutically acceptable,efficacious for their intended administration, and cannot degrade theactivity of the active ingredients of the compositions.

The amount of activating agent (TGF-αs or EGFs) present depends directlyupon the amount of TGF-βs present in the activated compositions of thisinvention. There are indications that the activation is not catalytic innature, and that therefore approximately stoichiometric (equimolar)quantities are preferred.

The amount of activated composition to be used in the methods of thisinvention cannot be stated because of the nature of the activity of TGFsand the nature of healing wounds and/or other traumata. As indicatedabove, the TGFs activate cells by binding to receptor sites on thecells, after which the TGFs are absorbed and utilized by the cells forthe synthesis of new protein, resulting in cell multiplication. Thus,the TGFs are consumed by the cell regenerating process itself, ratherthan acting in an enzymatic or other catalytic manner. Receptors forEGFs have been found on a wide variety of fibroblastic, epithelial, andparietal cells, as disclosed in Gonzalez et al, J. Cell. Biol.,88:108-144 (1981). Further, it has been calculated that there are 3,000EGF binding (receptor) sites for each rat intestinal epithelial cell, asdisclosed in M. E. Lafitte et al, FEBS Lett., 114(2):242-246 (1980). Itmust also be obvious that the amount of a cell growth promotingsubstance (such as the compositions of this invention) that must beutilized will vary with the size of the wound or other traumata to betreated.

Since the compositions of this invention both provoke and sustaincellular regeneration, a continual application or periodic reapplicationor the compositions is indicated.

The amount of active ingredient per unit volume of combined medicationfor administration is also very difficult to specify, because it dependsupon the amount of active ingredients that are afforded directly to theregenerating cells of the wound or other traumata situs. However, it cangenerally be stated that the TGF-βs should preferably be present in anamount of at least about 1.0 nanogram per milliliter of combinedcomposition, more preferably in an amount up to about 1.0 milligram permilliliter.

Additional Embodiments Utilizing the Compositions of this Invention

In addition to utilizing the activated TGF-β compositions of thisinvention by themselves, it is possible to use them in combination withsecondary growth factors.

The activated transforming growth factors of this invention may bephysically admixed with one or more of many other (secondary) peptideand nonpeptide growth factors. Such admixtures may be administered inthe same manner and for the same purposes as the activated transforminggrowth factors of this invention utilized alone to enhance theiractivity in promoting cell proliferation and repair.

The useful proportions of activated transforming growth factor tosecondary growth factors are 1:0.1-10 mols, with about equimolar amountsbeing preferred.

The secondary growth factors may be used alone or in any physiologicallyand pharmaceutically compatible combination.

The known secondary growth factors, in approximately descending order ofusefulness in this invention (by group), include:

1. platelet-derived growth factors.

2. fibroblast growth factors angiogenesis factors

3. insulin-like growth factors including somatomedins

4. insulin nerve growth factors

5. anabolic steroids.

In addition to the above known secondary growth factors, it isreasonable to expect that as yet undiscovered secondary growth factorswill be useful in admixture.

This invention also incorporates the inactive intermediate substanceTGF-β per se. Prior to this invention, this substance had not beenisolated or identified. TGF-β is believed to be substantially the sameor very similar for each animal species, regardless of the individual ofthat species or the particular body cells from which it is derived.Since TGF-β has been shown to be non-species-specific between rodents,cattle, and human beings, it is also reasonable to believe that thesubstance is substantially the same or very similar when derived fromany mammal, and possibly from any animal source. It should be noted,moreover, that this invention includes TGF-β regardless of the sourcefrom which it is isolated or derived, including genetically engineeredcells. It is well within the capabilities of biochemical technology togenetically engineer a cell to produce TGF-β at the present time.

Administration of Unactivated TGF-β

It is believed that TGF-β has no wound-healing or other tissue-repairactivity unless it has been activated by an agent as described above.

However, it is noted that Table 2 Experiment 5, supra, appears toindicate statistically similar results for TGF-β activated with EGF(chambers A, B, C) and TGF-β per se (chambers D, E, F). The most logicalexplanation for this, is that the TGF-β per se was activated by a TGFalready present in the test animal. Various TGFs, such as EGF, are knownto be present in blood plasma.

Thus, the results of Experiment 5 are not inconsistent with thisinvention, but instead constitute a variant embodiment thereof.Specifically, TGF-β per se may be administered, in accordance with thisinvention, instead of activated TGF-β, when there are sufficientendogenous activating agents present in an animal, to activate an amountof TGF-β sufficient to promote cell proliferation and tissue repair. Itis anticipated that in an animal suffering from the traumatacontemplated herein, there usually will not be sufficient endogenousactivating agents present.

The disclosures of the following applications, which were filed on thesame data as the present continuation-in-part application, i.e., June 3,1983, are entirely incorporated herein by reference:

1. "Transforming Growth Factor-beta From Human Platelets", by Richard K.Assoian, Charles A. Frolik, Michael B. Sporn and Anita B. Roberts, U.S.Ser. No. 500,832 (now abandoned).

2. "Transforming Growth Factor-beta From Human Placentas", by Charles A.Frolik, Richard K. Assoian, Michael B. Sporn, and Anita B. Roberts, U.S.Ser. No. 500,927 (now abandoned).

EXAMPLE 6 TGF-β from Human Platelets (Ser. No. 06/500,832)

Transforming growth factors have been detected in a variety ofnon-neoplastic tissues, but major sites of storage have not beenidentified. However, a comparison of both specific activities in initialextracts and yields of purified TGF-β, shows that platelets are a majorstorage site for the growth factor; they contain 40-100 fold more TGF-βthan do the other non-neoplastic tissues which have been examined. Thisfinding, in conjunction with the known role of platelets in woundhealing, supports the hypothesis that at least one physiological role ofTGF-β is to facilitate tissue repair and regeneration.

The total purification of platelet-derived TGF-β was facilitated by boththe high specific activity of the platelet extract and the aberrantelution of the polypeptide during gel filtration. Contaminants withmolecular weights similar to a column of the TGF (25,000 daltons) wereremoved on acrylamide gel in 1M acetic acid. In this system TGF-β eluteswith proteins of half its mass. (An apparent discrepancy in onefraction--high biological activity and no detectable protein at 25,000daltons--was due to the fact that detection of TGF-β by bioassay is atleast 100-fold more sensitive than chemical detection of the protein byelectrophoresis and silver staining). Addition of urea to the eluantprevented this retardation and resulted in the complete separation ofTGF-β from the lower molecular weight peptides. The overall recovery ofbiological activity from the purification procedure is somewhat low(about 5%), but control studies showed that other platelet factorsmodulate TGF-β action. (The specific activity of TGF-β decreased atleast 10-fold when it was assayed in medium containing 10%plasma-derived rather than whole-blood derived serum; data not shown).Removal of these factors during the purification procedure may wellexplain the observed decreases in total TGF-β biological activity. Themaximal biological activity of PDGF also requires the presence of otherbio-active peptides.

Purified, platelet-derived TGF-β was characterized chemically andbiologically (Tables 4 and 5). Its molecular weight (25,000 daltons),subunit structure (two 12,500-dalton polypeptides indistinguishable bySDS-polyacrylamide gel electrophoresis), and amino acid compositiondiffer from that of PDGF. Moreover, PDGF is a potent mitogen whereasplatelet-derived TGF-β is, at best, weakly mitogenic. Using similarbiochemical criteria platelet-derived TGF-β is also distinct from theplatelet protein family comprised of CTAP-III, β-thromboglobulin, andplatelet factor 4.

The role of platelets as a source of growth factors has receivedwidespread attention since the identification of PDGF. Aplatelet-derived peptide (C-TAP III; 9300 daltons) has been purified tohomogeneity and shown to be mitogenic for connective tissue cells. Twoplatelet growth factors distinct from PDGF have been identified on thebasis of their isoelectric points. Recently, it has been shown that TGFactivity is present in platelets and that the activity is enhanced byEGF. These studies with partially purified preparations yielded twoactive components during gel filtration (M_(r) =12-16,000 and 6,000).The larger protein is likely the 25,000 dalton TGF-β described hereineluting with an aberrantly low molecular weight during gel filtration inthe absence of denaturant. The smaller TGF was not detected butattention has been focused only on the most active TGF species inplatelets. Transforming growth factors having specific activities lessthan 10% of that of the 25,000-dalton TGF-β would not be detected withthe activity limits imposed herein.

Studies implicating PDGF in atherosclerosis and control of cell divisionhave emphasized its physiological release from platelets during theiraggregation at a site of injury. However, the characterization of PDGFas a competence factor suggests that platelet-mediated control of cellgrowth likely involves a complex synergism between several bio-activepeptides. Platelet-derived TGF-β has strong growth promoting ability,but it is not a strong mitogen. This unusual combination of biologicalproperties suggests that this protein may play a unique role in thosephysiological and pathological processes where platelet-derived factorsmodulate cell proliferation.

Examples of Platelet Derived TGF-β Purification and Analysis

Platelet Extraction: Platelet concentrates (20-30 units, 2-5 days old)were obtained through the courtesy of the National Institutes of HealthBlood Bank (Bethesda, Md., U.S.A.) and centrifuged (3200×g, 30 min.) toremove remaining plasma proteins. The platelets were washed twice bysuspension in 500-ml portions of Tris-HCl/citrate buffer, pH 7.5, andcentrifugation as described above. Washed platelets (20-30 g wetweights) were added to a solution of acidic ethanol prepared asdescribed elsewhere and immediately extracted in a homogenizer (4 mlacidic ethanol per g platelets). After incubation overnight at 4° C.,precipitated proteins were removed by centrifugation, and the resultingsupernatant was adjusted to pH 3 by addition of NH₄ OH. Proteins and TGFactivity were precipitated from the solution (overnight at 4° C.) byaddition of ethanol (2 vol, 0° C.) and ethyl ether (4 vol, 0° C.). Theprecipitate was collected by centrifugation and suspended in 1M aceticacid (10 ml). TGF activity was solubilized during an overnightextraction at 4° C. Centrifugation clarified the solution; thesupernatant was freeze-dried or subjected directly to gel filtration.The amount of protein in the extract was determined by weight or byreaction with Coomassie Blue using bovine plasma albumin as reference.

Purification of Platelet-Derived TGF-β: The solubilized platelet extract(10 ml in 1M acetic acid) was gel-filtered at a flow rate of 20 ml/h ona column (4.4×115 cm) of acrylamide gel equilibrated in 1M acetic acid.Fractions containing 5 ml were collected. The elution position of TGF-βwas determined by bioassay as described below, and the fractionscontaining the peak of activity were pooled and freeze-dried. The amountof protein in the pool was determined as described above. The residuewas dissolved in 0.5 ml of 1M acetic acid containing 8M ultra-pure ureaand gel-filtered at a flow rate of 3 ml/h on a column (1.6×85 cm) ofacrylamide gel which had been equilibrated in the sample solvent.Fractions containing 0.5 ml were collected. (To preclude the formationof cyanate in the solvent, the ultra-pure urea was dissolved at pH 2 in1M acetic acid. The resulting solution was adjusted to final conditionsby addition of glacial acetic acid and water). Aliquots of selectedcolumn fractions were tested for TGF-β activity. Fractions containingthe peak of TGF-β activity were pooled, dialyzed against 1M acetic acidto remove urea, and quick-frozen for storage at -20° C. The amount ofTGF-β in the final solution was determined by amino acid analysis (seebelow).

Bio-assay of TGF-β: The bioassay of TGF-β determines the ability of thepolypeptide to induce anchorage-independent growth in non-neoplasticNRK-fibroblasts by measuring the formation of colonies of cells in softagar. The assay was performed as described in Roberts et al, Proc. Nat.Acad. Sci. U.S.A., 77:3494-3498 (1980) except that 1) 3500 cells wereused per dish, 2) incubation proceeded for 7 days at 37° C. in ahumidified atmosphere of 10% CO₂ in air, and 3) TGF-β activity wasdetermined in the presence of EGF (2.5 ng/ml). Samples were sterilizedin 1M acetic acid and freeze-dried in the presence of bovine serumalbumin (100 μg) as carrier prior to analysis. Stained colonies werequantitated by number and size. One unit of TGF-β activity is defined asthat biological response resulting in 50% of maximal colony formation(colony size>3000 μm²) in the presence of Epidermal Growth Factor (EGF)(2.5 ng/ml). The maximal response of the assay is about 2500 colonies(>3000 βm²) per dish.

Mitogen Assay: NRK-fibroblasts were suspended in medium (Dulbecco'sModified Eagles Medium supplemented with 100 units per ml penicillin and100 μg per ml streptomycin), 10% in calf serum. Cells (4×10³ in 0.1 ml)were seeded in 96-well microtitre plates and incubated overnight. (Allincubations proceeded at 37° C. in a humidified atmosphere of 10% CO₂ inair). The resulting monolayers were washed twice with 0.2-ml portions ofserum-free medium and once with 0.2 ml of medium containing 0.2% calfserum. DME, 0.2% in calf serum (100 μl), was added to the washedmonolayers. The cells were incubated for 3-4 days during which time theyreached about 75% confluency. Test samples (50 μl, freeze-dried from 1Macetic acid and redissolved in 20 μl of sterile 4 mM HCl and 40 μl ofserum-free medium) were added to the growth arrested cells. Afterincubation (17 h), .sup. 3 H-thymidine (80 Ci/mmol) was added (1 μCi in50 μl of serum-free medium). Four hours later the medium was removed,and the cells were fixed (10 min at 4° C.) with ice-cold 5%trichloroacetic acid (0.2 ml). Fixed cells were washed 4 times with0.2-ml portions of 5% trichloroacetic acid. Precipitated radioactivitywas solubilized by incubation in 0.5M NaOH (0.15 ml per well for 30 minat 37° C.).

RESULTS AND INTERPRETATION

The biological properties of purified, platelet-derived TGF-β are shownbelow in Table 4. In the presence of EGF, the TGF elicits near maximaltransforming activity at a concentration of 1 ng/ml. In agreement withthe data of others using impure TGF-β, the activity of the growth factoris destroyed by reduction; stimulation of colony formation by anEGF/reduced TGF-β mixture was no greater than the EGF alone. Moreover,TGF-β, assayed in the absence of EGF, gave the basal level (shown by 10%calf serum) of transforming activity. Other experiments showed thatTGF-β (1 ng/ml) does not compete for the binding of ¹²⁵ I-labeled EGF tothe EGF receptor.

TGF-β can be detected in the platelet extract at protein concentrationsshowing no mitogenic activity. Table 4 shows that purified TGF-β (1ng/ml) does not stimulate ³ H-thymidine incorporation intoNRK-fibroblasts despite the fact that these cells respond to establishedmitogens. [Decreased ³ H-thymidine incorporation, relative to basal, wasobserved with TGF-β when used at concentrations greater than 0.1 ng/ml.At no concentration tested (0.01-10 ng/ml) did the TGF stimulate ³H-thymidine incorporation]. In addition to confirming thatplatelet-derived TGF-β is biologically distinct from PDGF, these datasuggest that the role of TGF-β in inducing cell growth in soft agar maybe unrelated to a direct stimulation of total DNA synthesis.

The sensitivity of TGF-β to treatment with dithiothreitol (Table 4)indicates that disulfide bonds likely play an important role inconferring structure to the molecule. The molecular weight ofplatelet-derived TGF-β, as determined by SDS-polyacrylamide gelelectrophoresis, is affected by treatment with reductant. This resultindicates that the native protein (M_(r) =25,000) is composed of twopolypeptide chains of very similar molecular weight (M_(r) =12,500)which are maintained in covalent association by disulfide bonds. (Theinability to detect contaminants in the presence as well as absence ofreductant further confirms the purity of the protein).

                  TABLE 4                                                         ______________________________________                                        Biological effects of purified platelet-derived                               TGF-β                                                                                  Number of  Amount of                                                          Colonies   .sup.3 H-Thymidine                                   Sample        (>3000 μm.sup.2)                                                                      Incorpration (CPM)                                   ______________________________________                                        TGF-β & EGF                                                                            1980       ND                                                   reduced TGF-β                                                                           380       ND                                                   & EGF                                                                         EGF            400       45,200                                               TGF-β     25         4,800                                               calf serum (10%)                                                                             12        86,000                                               ______________________________________                                    

This table shows the biological properties of purified TGF-β at aconcentration of 1 ng/ml (a concentration 10-fold greater than thatyielding 50% of maximal transforming activity). EGF was used at 2.5ng/ml, its concentration in the TGF-β bioassay. The growth factors weredissolved in 1M acetic acid with 10 μg BSA as carrier and freeze-driedprior to analysis. To prepare reduced TGF-β, the lyophilized growthfactor and BSA carrier were treated with a molar excess ofdithiothreitol (0.05M in 0.2 ml of 0.1M sodium phosphate buffer, pH 7.4;3 h at 37° C.). The solution of reduced TGF-β was acidified with aceticacid (40 μl), dialyzed against 1M acetic acid in a microdialysis unit,and freeze-dried prior to analysis. EGF was added to the sample afterdialysis. A mock reduction (performed in the absence of dithiothreitol)had no effect on TGF-β transforming activity. In the mitogen assay, thebasal level of ³ H-thymidine incorporation (determined in the absence ofmitogen) was 9000-10,000 CPM. The mitogenic activity of TGF-β was notdetermined (ND) in the presence of EGF.

TABLE 5

Partial Amino Acid Sequence of Platelet-derived TGF-β

                  TABLE 5                                                         ______________________________________                                        Partial Amino Acid Sequence of Platelet-derived                               TGF-β                                                                     ##STR2##                                                                     ______________________________________                                    

(where X is undetermined) as determined by Edman Degradation, eachsubunit probably having the same above sequence.

EXAMPLE 7 TGF-β From Human Placenta (Ser. No. 06/500,927)

The acid-ethanol extract of human placenta displayed activity thatstimulated anchorage-dependent NRK cells to form colonies in soft agar.EGF markedly enhanced (150 fold) the activity of this placental TGF. Ashas been previously demonstrated for other TGFs, the activity of apartially purified placental preparation was destroyed by treatment witheither trypsin or dithiothreitol.

Chromatography of the undialyzed crude residue from the combinedacid-ethanol extractions of 11 placentas on a column in 1M acetic acidgave two peaks of activity when assayed in the presence of EGF (pool A,apparent M_(r) 5000-9000 and pool C, apparent M_(r) less than 3500). Nocolony stimulating activity was detected when equivalent aliquots wereassayed in the absence of EGF. Therefore, all subsequent soft agarassays were performed in the presence of 2 ng/ml EGF. None of the 3pools competed with ¹²⁵ I-EGF for EGF membrane receptor sites on CCL-64cells. This placenta-derived TGF is therefore clearly a member of theTGF-β family. Pool A, which contained 47% of the recovered protein, had17% of the recovered TGF activity (see Table 6) while pool C, with only3.3% of the protein, contained 18% of the recovered activity. Pool B didnot give a valid assay for TGF activity because of the presence of agrowth inhibitory substance. This inhibitor could be separated from thesoft agar colony forming activity by further chromatography. Asindicated in Table 6, 69% of the TGF activity found in the crude residuewas present in the pool B fraction that eluted from the column. Pools Band C were therefore used for further purification.

Application of the protein from the gel filtration column to acation-exchange column and subsequent elution of the applied materialwith a linear sodium chloride gradient, gave a single peak of soft agarcolony forming activity. Although 85-96% of the applied protein wasrecovered from the column, only 10-45% of the applied TGF activity wasdetected. Whether this loss of activity is due to specific loss of theTGF protein, to denaturation of the TGF, or to the separation of the TGFfrom an activator is, at this time, still under investigation. Fractionswere pooled and chromatographed on HPLC column using anacetonitrile-0.1% TFA gradient. The TGF activity for both pools elutedfrom the column as a single peak at an acetonitrile concentration of35%. Rechromatography of this material on a CN support equilibrated inn-propanol-0.1% TFA yielded a single peak of TGF activity at 35%n-propanol which corresponded to a strong absorbance peak. Thehomogeneity of the final preparation was indicated by gelelectrophoresis. The final degree of purification of placenta derivedTGF-β from the crude extract was 110,000-124,000 fold with a 1.1%recovery of activity in pool C and 4.8% in pool B. Only 64-72 pg/ml ofplacental TGF-β was needed to obtain a half-maximal growth stimulatoryresponse (ED₅₀) in the presence of 2 ng/ml of EGF.

The purity of the final TGF preparation was also demonstrated byNaDodSO₄ -polyacrylamide gradient gel electrophoresis. In the absence ofβ-mercaptoethanol, a single polypeptide band with an apparent molecularweight of 23,000-25,000 was observed for TGF from either pool B or poolC. Reduction of the protein with β-mercaptoethanol produced a singleband at approximately 13,000 molecular weight. When the gel was slicedinto 0.5 cm strips and the unreduced protein eluted into 1M acetic acid,all the TGF activity was found in the slice that corresponded to amolecular weight of 23,000-25,000, clearly indicating that the TGFactivity corresponded to the only detectable protein band.

Examples of Extraction, Purification, and Analysis

Soft Agar Assay--Soft agar colony forming activity was determined asdescribed previously except that the cells were stained at the end ofone week in assay and the number and size of the colonies weredetermined using image analysis system.

Extraction--Normal term human placentas were frozen on dry ice within 30minutes after delivery and stored at -60° C. until used. Placentas wereextracted using a procedure previously described in Roberts et al, Proc.Nat. Acad. Sci. USA, 77:3494-3498 (1980), except that the homogenizedtissue (600-1000 g) was stirred in the acid-ethanol solution at roomtemperature for 2 to 3 hrs prior to centrifugation. The resultingsupernatant was adjusted to pH 3.0 and protein precipitated with etherand ethanol. The precipitate was collected by filtration and redissolvedin 1M acetic acid (1 ml/g of tissue). Insoluble material was removed bycentrifugation, the supernatant lyophilized and the residue (27 mg pergram wet weight placenta) stored at -20° C.

Gel filtration chromatography--The lyophilized extract (239 g) from 11placentas (8.8 kg) was redissolved in 1M acetic acid (50 mg residue perml) and applied in two separate portions (107 g and 132 g residue) to acolumn (35.6×90 cm) containing acrylamide gel (100-200 mesh),equilibrated and eluted (1.6 L/hr) with 1M acetic acid at roomtemperature. Fractions (800 ml) were collected and aliquots of the evennumbered fractions were assayed for protein and for growth promotingactivity in soft agar. The fractions containing TGF activity werecombined into three separate pools (A-C) and lyophilized. Pool B (6 gresidue per column) was redissolved in 1M acetic acid (60 mg/ml) andapplied to a column (10×91 cm) containing P-6 acrylamide gelequilibrated with 1M acetic acid. The protein was eluted from the columnwith 1M acetic acid (150 ml/hr), collecting 37 ml fractions. Aliquots ofeven numbered fractions were assayed for TGF activity. The fractionscontaining this activity were pooled and lyophilized.

Ion-Exchange Chromatography--Twenty-four percent of pool B from the P-6column (2.1 g protein) and pool C from the P-30 column (1.9 g protein)were redissolved separately in 60 ml 0.01M acetic acid. The pH wasadjusted to 4.5 and the conductivity to 1.2-1.5 mS/cm. Each sample wasthen applied to a cation exchange column (CM-Trisacryl M, LKB, 5×10 cm)equilibrated in 0.05M sodium acetate, pH 4.5 (buffer A). The column waseluted with 300 ml of buffer A (145 ml/hr) followed by a linear sodiumchloride gradient to 0.70M sodium chloride in buffer A at 0.8 mM/min.After 70 fractions (29 ml/fraction), the column was washed with 1Msodium chloride, 0.05M sodium acetate, pH 2.5 and then reequilibratedwith buffer A. Aliquots from the even numbered fractions were removedfor determination of protein and TGF activity. The peak of activity wascombined for further analysis.

Reverse-Phase HPLC--The sample from the ion-exchange column was made 10%(v/v) in acetonitrile, 0.1% (v/v) in trifluoroacetic acid (TFA) and thepH adjusted to 2.0. It was then pumped onto an HPLC column (10 μmparticle size, 0.78×30 cm) equilibrated in acetonitrile:water:TFA(10:90:0.1), pH 2. After washing the sample onto the column with 50 mlof the initial solvent, the column was eluted (1.2 ml/min) with a 60 minlinear gradient from 25:75:0.1 to 45:55:0.1 acetonitrile:water:TFA, pH2. After 75 fractions (1.2 ml/fraction) the column was stripped withacetonitrile:water:TFA (80:20:0.1), pH 2, collecting 2.4 ml fractions.Aliquots (5 μl) were removed for assay of TGF activity.

The peak of TGF activity from the HPLC column was combined, lyophilized,redissolved in n-propanol:water:TFA (30:70:0.1), pH 2, and applied to aCN column (10 μm particle size, 0.38×30 cm) equilibrated with the samplesolvent. The column was then eluted (1.1 ml/min) with a 153 min lineargradient from 30:70:0.1 to 45:55:0.1 n-propanol:water:TFA, pH 2.Forty-five fractions (2.2 ml/fraction) were collected and aliquots wereremoved for bioassay, amino acid analysis, and gel electrophoresis.

NaDodSO₄ -Polyacrylamide Gel Electrophoresis--Samples were analyzed on1.5 mm slab gels using either a polyacrylamide gradient of 15 to 28% ora 15% polyacrylamide gel and a discontinuous buffer system. Proteinswere fixed with formaldehyde and stained using a silver stainingtechnique. In some cases, samples were boiled with 5% β-mercaptoethanolfor 3 min prior to application to the gel.

Other procedures--Total protein was determined either by the dye-bindingmethod or fluorescamine assay using bovine serum albumin as standard orby amino acid analysis. Assays for EGF-competing activity were performedas previously described.

A summary of a general extraction procedure found to be preferable topreviously used procedures, is given below.

Example of a Placenta Derived TGF-β Extraction Protocol

1. Placentas are placed on dry ice immediately after delivery and arestored at -70° C. or colder until used.

2. Approximately 24 hr before extraction, thaw placentas at -20° C.

3. Chop 1 kg of placenta into pieces and place into extraction solution(4 L solution/kg tissue).

Extraction Solution:

3189 ml 95% ethanol

770 ml water

66 ml concentrated HCl

210 mg phenylmethylsulfonyl fluoride

12 mg pepstatin A

4. Mince in a blender to give a slurry.

5. Stir slurry at room temperature for approximately 21/2 hr (requiresheavy-duty stirrer).

6. Centrifuge--17,700×g--10 min.; Discard pellet, save supernatant.

7. Adjust the supernatant to pH 3.0 with concentrated ammoniumhydroxide.

8. Add 0.55 volume to 5.5M NaCl.

9. Precipitate overnight at 4° C.

10. Centrifuge--17,700×g--10 min. Discard pellet, save supernatant.

11. Concentrate supernatant to 1/5 volume or less. (We used a hollowfiber concentrator with a 5000 MW nominal cutoff membrane).

12. Add 2 volumes 5.5M NaCl to concentrated supernatant.

13. Precipitate overnight at 4° C.

14. Centrifuge--17,700×g--10 min. Discard supernatant. Save pellet forgel filtration chromatography and further purification.

                                      TABLE 6                                     __________________________________________________________________________    Purification of TGF-β from human placenta.                                                         Total Degree                                                                            Recovery                                           Protein*   Specific.sup.++                                                                     activity                                                                            of puri-                                                                          of                                        Purification                                                                           recovered                                                                           ED.sub.50+                                                                         activity                                                                            (units ×                                                                      fication                                                                          activity                                  step     (mg)  (ng/ml)                                                                            (units/ug)                                                                          10.sup.3)                                                                           (fold)                                                                            (%)                                       __________________________________________________________________________      Crude  239,000                                                                             7,600                                                                              0.09  21,510                                                                              1.0 100                                         Extract                                                                       Acrylamide                                                                    Gel #1                                                                        Pool A 73,900                                                                              15,000                                                                             0.05  3,695 0.6 17                                          Pool B 27,720                                                                              --   --    --    --  --                                          Pool C 1,900 360  2.0   3,800 22  18                                          Acrylamide                                                                    Gel #2                                                                        Pool B 8,700 410  1.7   14,790                                                                              19  69                                          Ion-Exchange                                                                  Pool B§                                                                         140   62   11.5  1,610 128 31                                          Pool C 46.3  85   8.4   390   93  1.8                                         HPLC-C.sub.18                                                                 Pool B 0.27  0.10 7,000 1,900 77,000                                                                            37                                          Pool C 0.26  1.2  595   155   6,610                                                                             0.7                                         HPLC-CN                                                                       Pool B 0.025 0.072                                                                              9,920 248   110,000                                                                           4.8                                         Pool C 0.022 0.064                                                                              11,160                                                                              245   124,000                                                                           1.1                                       __________________________________________________________________________

Table 6, above, summarizes the examples and results of the purification.

                  TABLE 7                                                         ______________________________________                                        Partial Amino Acid Sequence For Each Of The Two                               Human Placenta TGF-β Subunits.                                           (CMC is half-cystine or cysteine, determined as S-                            carboxymethylcysteine).                                                        ##STR3##                                                                      ##STR4##                                                                      ##STR5##                                                                     where X is undetermined.                                                      ______________________________________                                    

SUMMARY AND DISCUSSION OF RESULTS

A TGF has been isolated from the acid-ethanol extract of human placenta.It is classified as a type β TGF, because it does not compete with EGFfor membrane receptor sites but requires EGF for the induction of colonygrowth in soft agar, with a 50% maximal formation of colonies greaterthan 60 μm diameter occurring at 64-72 pg TGF per ml (3×10⁻¹² M). Thefactor has been purified to homogeneity by gel filtration,cation-exchange and high-pressure liquid chromatography. It is a proteinof molecular weight 23,000 to 25,000 and is composed of two polypeptidechains of approximately 13,000 molecular weight held together bydisulfide linkages. Whether these chains are identical or differentremains to be determined. Although the protein contains 16 half-cystineresidues, it is not yet known whether all of these residues are involvedin disulfide linkage. However, the extreme stability of the TGFs to acidtreatment and heat denaturation suggests the presence of a large numberof such bonds.

The presence of TGFs in the crude acid-ethanol extract of human placentathat were able to compete with EGF for binding to membrane receptors(TGF-αs) has recently been noted in Stromberg et al, Biochem. Biophys.Res. Commun., 106: 354-361 (1982). In the present invention, asignificant amount of TGF-α-like activity was not detected. Althoughsome soft agar colony forming activity was found in the crude residue inthe absence of EGF, this activity did not compete with EGF in a receptorbinding assay and it was stimulated 150 fold by the addition of 2 ng/mlof EGF, indicating that most, if not all, of the TGF present was of thetype β class. Also, as the placental TGF was purified, it became totallydependent on exogenous EGF for soft agar colony forming activity. Partof this difference may be explained by the fact that in Stromberg et al(1982) colonies of 6 cells or greater were considered to be significantwhile for the present invention colonies had to contain at least 60cells in order to be counted.

A TGF-β at a concentration of 430 ng per gram wet weight of tissue hasrecently been purified from human platelets. Because placenta containsmuch blood, it is possible that the placental TGF-β (10 ng per gram oftissue) originated from the platelets. However, even assuming that theplacenta was 100% blood and that platelets comprised 0.2% of this blood,platelet TGF would account for only 8% of the recovered placental TGF.Therefore, if the placental TGF β did originate from the platelets, itwould have to be concentrated by an, as yet, unknown mechanism.

Blood platelets also contain the peptide, platelet derived growth factor(PDGF). However, placental TGF-β is not PDGF, as clearly demonstrated bythe results from two different assays. In the first assay, placentalTGF-β did not have any chemotactic activity when tested under conditionswhere PDGF displayed strong activity. Similarly, placental TGF-β did notcompete with PDGF in a radioreceptor assay.

Although TGFs were originally found in tumor cells and were postulatedto be involved in transformation and neoplastic cell growth, theirpresence in adult cells and tissues, in platelets, and in embryos [asreported in Twardzik et al, Cancer Res., 42: 590-593 (1982)] imply thatTGFs have a normal physiological function as well. The purification ofplacental TGF-β to homogeneity facilitates investigation of thisfunction, since it permits the development of both receptor binding andradioimmunoassays. These assays not only allow a specific, quickprocedure for quantitation of TGF-β but will also permit investigationof the mechanisms of action and the control of expression of TGF-βsunder normal and neoplastic conditions. Finally, structural analysis ofpurified TGF-β provides information for initiation of cloningexperiments. This will allow eventual production of large quantities ofhuman TGF-β, which might have useful therapeutic applications inenhancement of wound healing and tissue repair.

What is claimed is:
 1. Isolated and substantially homogeneous betatransforming growth factor (TGF-β) having the followingcharacteristics:acid stable; an apparent molecular weight of about25,000 daltons in the absence of a reducing agent as measured bySDS-PAGE; an apparent molecular weight of about 12,500 daltons underreducing conditions as measured by SDS-PAGE; does not compete withepidermal growth factor for receptor binding; induces dose-dependentformation of large colonies having a size of greater than 3,100 μm² ofNRK 49F cells in a soft agar assay when activated by epidermal growthfactor (EGF) or transforming growth factor-alpha (TGF-α); and beingpurified to the extent that(1) it does not itself induce NRK cells toform said large colonies in soft agar and (2) a single band with anapparent molecular weight of about 12,500 daltons is shown on SDS-PAGEunder reducing conditions and a single band with an apparent molecularweight of about 25,000 is shown under non-reducing conditions. 2.Isolated and substantially homogeneous beta transforming growth factor(TGF-β) having the following characteristics:acid stable; an apparentmolecular weight of about 25,000 daltons in the absence of a reducingagent as measured by SDS-PAGE; an apparent molecular weight of about12,500 daltons under reducing conditions as measured by SDS-PAGE; doesnot compete with epidermal growth factor for receptor binding; inducesdose-dependent formation of large colonies having a size of greater than3,000 μm² of NRK 49F cells in a soft agar assay in the presence ofepidermal growth factor (EGF); and being purified to the extent that(1)it does not itself induce NRK cells to form said large colonies in softagar and (2) a single band with an apparent molecular weight of about12,500 daltons is shown on SDS-PAGE under reducing conditions and asingle band with an apparent molecular weight of about 25,000 is shownunder non-reducing conditions, wherein said TGF-β has the followingpartial amino acid sequence in a subunit thereof as determined by Edmandegradation: Ala-Leu-Asp-Thr-Asn-Tyr-X-Phe-Ser-, where X isundetermined.
 3. The TGF-β of claim 1, which elicits 50% of its maximalbiological response at concentrations less than 5×10⁻¹² M.
 4. Isolatedand substantially homogeneous beta transforming growth factor (TGF-β)having the following characteristics:acid stable; an apparent molecularweight of about 23,000-25,000 daltons in the absence of a reducing agentas measured by SDS-PAGE; an apparent molecular weight of about 13,000daltons under reducing conditions as measured by SDS-PAGE; does notcompete with epidermal growth factor for receptor binding; inducesdose-dependent formation of large colonies having a size of greater than3,000 μm² of NRK 49F cells in a soft agar assay when activated byepidermal growth factor (EGF); and being purified to the extent that(1)it does not itself induce NRK cells to form said large colonies in softagar and (2) a single band with an apparent molecular weight of about13,000 daltons is shown on SDS-PAGE under reducing conditions and asingle band with an apparent molecular weight of about 23,000-25,000 isshown under non-reducing conditions, wherein said TGF-β has thefollowing partial amino acid sequence in a subunit thereof as determinedby Edman degradation: ##STR6## where CMC is Half-cystine or cysteine,determined as S-carboxymethylcysteine and X is undetermined and whereinsaid TGF-β causes half-maximal growth of NRK indicator cells in softagar at about 64-72 pg/ml in the presence of 2 ng/ml of EGF.